Abstract

This chapter discusses the quantitative microscopy of green fluorescent protein-labeled yeast. With the development of methods to tag proteins using green fluorescent protein (GFP), fluorescence microscopy has become increasingly important for characterizing protein function in yeast. The chapter describes the use of three-dimensional (3-D) deconvolution microscopy to perform fixed- and live-cell analysis of cells carrying GFP-tagged proteins. The chapter also discusses the process of deconvolution in fluorescence microscopy. Despite the focus on high-performance imaging, the methods described in the chapter are applicable to a wide range of experiments using conventional wide-field microscopes. The chapter describes the various steps for preparing biological samples, such as strain construction, mounting cells for microscopy, preparing slides with agar pads for live-cell microscopy, mounting cells on agar pads, mounting live cells without agar pads, fixing cells with paraformaldehyde and mounting, optimizing microscope optics, selecting filters, selecting cover glass, temperature control, minimizing spherical aberration through oil matching, acquiring images, and viewing and printing the image.

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